The objective of the proposed work is to understand how the Dmrt1 gene controls development and function of the testis. The testis has two essential functions: production of sperm, the cells that serve as vehicles for the immortality of male germ line DNA;and production of hormones that direct other parts of the body to develop in a male-specific manner. We discovered the Dmrt family of conserved transcriptional regulators and have shown that several DMRT proteins are important for gonadal development in vertebrates. Loss of DMRT1 causes severe defects in testis development and, in humans, is associated with male-to- female sex reversal. Recently we found that Dmrt1 is required in the 129/Sv mouse strain to control germ cell pluripotency, preventing these cells from forming other cell types of the body rather than sperm. The main hypothesis of this proposal is that Dmrt1 plays a central role in controlling germ line stem cells, acting at different stages both in the stem cells themselves and in surrounding support cells called Sertoli cells. We have four aims. Aim 1 asks how DMRT1 controls germ cell pluripotency in the embryo. We will identify changes in Dmrt1 mutant embryonic germ cells as they lose their normal fate commitment, determine whether Dmrt1 controls this process in the germ cells or the Sertoli cells, and identify candidate regulators of germ cell pluripotency. Aim 2 tests the function of DMRT1 during adult spermatogenesis, focusing on adult germ line stem cells. We will use an innovative genetic strategy to determine the role of Dmrt1 in adult germ line stem cells. Aim 3 seeks to illuminate the function of DMRT1 by identifying the genes it directly regulates in the neonatal testis. Aim 4 tests the hypothesis that Dmrt1 acts with the adjacent Dmrt3 gene to coordinately direct embryonic testis development, assessing the phenotype of Dmrt3 and Dmrt1Dmrt3 double mutants on sensitized genetic backgrounds. This will provide a mouse model for human sex-reversing deletions that remove the two genes. The work we propose is highly relevant to human health. DMRT1 is implicated in human infertility, in testicular dysgenesis, and in testicular cancer. Our studies may permit better diagnosis and treatment of these conditions. Furthermore, because Dmrt1 plays a role in controlling germ line pluripotency, our work may aid in the use of germ line stem cells for therapeutic cloning and for restoration of fertility after chemotherapy.